The unifying hypothesis of the program project is that altered glycolytic flux in diabetes results in the dysfunctional regulation of calcium- independent phospholipase A2 in the heart and blood vessels. This project examines the role of the dysfunctional regulation of the glycolytically-coupled generation of lipid second messengers by calcium- independent phospholipase A2 in the pathogenesis of contractile dysfunction in diabetic myocardium and the increased susceptibility of diabetic myocardium to acute ischemia. We have demonstrated that diabetic myocardium exhibits three hallmarks of pathologic alterations in lipid metabolism including: 1) dramatic depletion of the arachidonic acid content present in its endogenous phospholipid storage depots; 2) markedly increased levels of calcium-independent phospholipase A2 activity; and 3) abnormal fluxes of arachidonic acid into and out of myocytic phospholipid pools. Accordingly, Specific Aim 1 will examine the importance of the coupling of glycolytic flux with arachidonic acid release and lysophospholipid generation in diabetic myocardium. The role of calcium-independent phospholipase A2 in this process will be determined by exploiting the specificity inherent in the mechanism-based inhibitor (E)-6-(bromomethylene)-3-(1-naphthalenyl)-2-H-tetrahydropyran- 2-one(BEL). Next, the role of accelerated membrane hydrolysis by calcium-independent phospholipase A2 in mediating the increased susceptibility of diabetic myocardium to ischemic injury will be explored. In Specific Aim 2, the heterogeneity of arachidonic acid depletion in myocardial subcellular membrane compartments in diabetic myocardium will be examined by exploiting the power and sensitivity of electrospray ionization mass spectrometry. Alterations of lipid flux in specific subcellular membrane loci in the diabetic state will be identified utilizing quantitative electron microscopic autoradiography and the role of calcium-independent phospholipase A2 in this process will be determined utilizing BEL. In Specific Aim 3, the mechanisms underlying the marked increase in calcium-independent phospholipase A2 activity in diabetic myocardium will be identified by analysis of alterations in calcium-independent phospholipase A2 mass, isoform composition and/or post-translational modifications in the diabetic state. Collectively, these studies represent a multidisciplinary approach targeted at identifying the importance of glycolytic coupling of lipid second messenger generation to cardiac dysfunction in the diabetic state.